Assimilable organic carbon (AOC) determination using GFP-tagged Pseudomonas fluorescens P-17 in water by flow cytometry

Abstract

One of the newly developed methods for Assimilable organic carbon (AOC) determination is leveraged on the cell enumeration by flow cytometry (FC) which could provide a rapid and automated solution for AOC measurement. However, cell samples staining with fluorescence dye is indispensable to reduce background and machine noise. This step would bring additional cost and time consuming for this method. In this study, a green fluorescence protein (GFP) tagged strain derived of AOC testing strain Pseudomonas fluorescens P-17 (GFP-P17) was generated using Tn5 transposon mutagenesis. Continuous culture of this mutant GFP-P17 showed stable expression of eGFP signal detected by flow cytometry without staining step. In addition, this GFP-P17 strain displayed faster growth rate and had a wider range of carbon substrate utilization patterns as compared with P17 wild-type. With this strain, the capability of a new FC method with no dye staining was explored in standard acetate solution, which suggests linear correlation of counts with acetate carbon concentration. Furthermore, this FC method with GFP-P17 strain is applicable in monitoring GAC/BAC efficiency and condition as similar trends of AOC level in water treatment process were measured by both FC method and conventional spread plating count method. Therefore, this fast and easily applicable GFP-P17 based FC method could serve as a tool for routine microbiological drinking water monitoring.

Coupling pathway prediction and fluorescence spectroscopy to assess the impact of auxiliary substrates on micropollutant biodegradation

“…For this purpose, KN65.2 was inoculated together with the Pseudomonas fluorescens sp. P17—a strain commonly used to determine assimilable organic carbon in water samples (Hammes & Egli, 2005; Tang et al, 2018; van der Kooij, 2002). This setup allowed us to investigate the influence of a competitor (P17) on mixed substrate biodegradation by KN65.2.…”

Assessing the substrate specificity of a micropollutant degrading strain: generalist or specialist?

“…A generalist commonly used in the eld of water supply is the strain Pseudomonas uorescens P17, which grows on various (aromatic) amino acids, carbohydrates, alcohols and organic acids, and is commonly used to measure AOC in drinking water. [33][34][35] Regarding OMP degraders, Novosphingobium species from different habitats, including the carbofuran (carbamate pesticide) degrader Novosphingobium sp. KN65.2, show a wide range of enzymes for degrading xenobiotic aromatic compounds.…”